Wang M et al. (2023) Stud Health Technol Inform "Expression and Purification of SMGL-1."

Wang M, Mei K

[Stud Health Technol Inform, 2023]

Recombinant protein expression is a crucial technique in biology, with E. coli being the most widely used expression system. However, due to growth pressure, the expression of large molecular weight proteins in E. coli has remained a challenging task. SMGL-1, a newly discovered protein in C. elegans with Rabin 8 function, plays an important role in biology. To better understand the function of SMGL-1, we first predicted and analyzed its protein structure and properties using artificial intelligence. We then conducted studies on its expression and purification. Through optimization of IPTG concentration and expression strains, we successfully expressed SMGL-1 in E. coli, providing guidance for the expression of large proteins in E. coli. Furthermore, we explored the purification of SMGL-1 using GST affinity chromatography, Nickel affinity chromatography, and ammonium sulfate precipitation methods, laying the foundation for future purification work on SMGL-1.

Wang M et al. (2024) Brain Sci "Cellular and Molecular Mechanisms Underlying Synaptic Subcellular Specificity."

Wang M, Shao Z, Fan J

[Brain Sci, 2024]

Chemical synapses are essential for neuronal information storage and relay. The synaptic signal received or sent from spatially distinct subcellular compartments often generates different outcomes due to the distance or physical property difference. Therefore, the final output of postsynaptic neurons is determined not only by the type and intensity of synaptic inputs but also by the synaptic subcellular location. How synaptic subcellular specificity is determined has long been the focus of study in the neurodevelopment field. Genetic studies from invertebrates such as <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) have uncovered important molecular and cellular mechanisms required for subcellular specificity. Interestingly, similar molecular mechanisms were found in the mammalian cerebellum, hippocampus, and cerebral cortex. This review summarizes the comprehensive advances in the cellular and molecular mechanisms underlying synaptic subcellular specificity, focusing on studies from <i>C. elegans</i> and rodents.

Wang M et al. (1996) West Coast Worm Meeting "COMPETENCE OF VPCS DURING C.ELEGANS VULVAL INDUCTION"

Sternberg PW, Wang M

[West Coast Worm Meeting, 1996]

During C. elegans vulval development, the vulval precursor cells (VPCs) forms a fixed spatial pattern of three kinds of cell fates: tertiary, tertiary, secondary, primary, secondary, tertiary. This process of pattern formation is induced by an inductive signal LIN-3 (an EGF-like molecule), which is produced by the anchor cell (AC) in the somatic gonad. Each VPC is competent to respond to LIN-3 signal from the AC and to adopt any of the three fates during late L2 to early L3. By ablating the AC at different times, Kimble found that the VPCs are able to adopt their correct fates if the AC is ablated after about one hour prior to their division. Therefore, the AC is no longer required after that time. The question remains whether the temporal competence window of the VPCs extends past the time when the AC is required, or whether LIN-3 is made by the AC early, but is not received by the VPCs until late. To examine this issue, we have used a transgenic line (syIs12), which expresses the EGF domain of LIN-3 under the transcriptional control of an hsp16 promoter, in a lin-3 loss-of-function background. We induced LIN-3 by heatshock at different times and found that the VPCs lose their competence to respond to LIN-3 signal at or about their first division. We can now use this assay to define what sets the end of the VPC competence. We have tested the effect of activated LIN-12. A gain-of-function mutation of lin-12 makes all VPCs become secondary independent of the LIN-3 signal. We have constructed syIs12 in a lin-3 loss-of-function, lin-12 gain-of-function background. After inducing LIN-3 by heatshock, we observe that VPCs can adopt primary fate even after their first division. We conclude that LIN-12 activity extends the window of VPC competence to respond to LIN-3. We envision that LIN-12 has a dual role: it promotes the secondary fate and inhibits the tertiary fate. Primary and secondary fates share common features (including stimulation of the cell cycle). LIN-12 might drive VPCs along what is at first a common pathway, and thus remain competent to respond to LIN-3 by becoming primary.

Wang M et al. (2014) Biochemistry "Protein arginine methyltransferase 5 catalyzes substrate dimethylation in a distributive fashion."

Thompson PR, Fuhrmann J, Wang M

[Biochemistry, 2014]

Protein arginine methyltransferase 5 (PRMT5) is a histone-modifying enzyme whose activity is aberrantly upregulated in various cancers and thereby contributes to a progrowth phenotype. Indeed, knockdown of PRMT5 leads to growth arrest and apoptosis, suggesting that inhibitors targeting this enzyme may have therapeutic utility in oncology. To aid the development of inhibitors targeting PRMT5, we initiated mechanistic studies geared to understand how PRMT5 selectively catalyzes the symmetric dimethylation of its substrates. Toward that end, we characterized the regiospecificity and processivity of bacterially expressed Caenorhabditis elegans PRMT5 (cPRMT5), insect cell-expressed human PRMT5 (hPRMT5), and human PRMT5 complexed with methylosome protein 50 (MEP50), i.e., the PRMT5-MEP50 complex. Our studies confirm that arginine 3 is the only site of methylation in both histone H4 and H4 tail peptide analogues and that sites distal to the site of methylation promote the efficient symmetric dimethylation of PRMT5 substrates by increasing the affinity of the monomethylated substrate for the enzyme. Additionally, we show for the first time that both cPRMT5 and the hPRMT5-MEP50 complex catalyze substrate dimethylation in a distributive manner, which is assisted by long-range interactions. Finally, our data confirm that MEP50 plays a key role in substrate recognition and activates PRMT5 activity by increasing its affinity for protein substrates. In total, our results suggest that it may be possible to allosterically inhibit PRMT5 by targeting binding pockets outside the active site.

Wang M et al. (2013) Biochemistry "Substrate specificity, processivity, and kinetic mechanism of protein arginine methyltransferase 5."

Thompson PR, Xu RM, Wang M

[Biochemistry, 2013]

Protein arginine methyltransferases (PRMTs) have emerged as attractive therapeutic targets for heart disease and cancers. PRMT5 is a particularly interesting target because it is overexpressed in blood, breast, colon, and stomach cancers and promotes cell survival in the face of DNA damaging agents. As the only known member of the PRMT enzyme family to catalyze the formation of mono- and symmetrically dimethylated arginine residues, PRMT5 is also mechanistically unique. As a part of a program to characterize the mechanisms and regulation of the PRMTs and develop chemical probes targeting these enzymes, we characterized the substrate specificity, processivity, and kinetic mechanism of bacterially expressed Caenorhabditis elegans PRMT5 (cPRMT5). In this report, we demonstrate that distal positively charged residues contribute to substrate binding in a synergistic fashion. Additionally, we show that cPRMT5 catalyzes symmetric dimethylation in a distributive fashion. Finally, the results of initial velocity, product, and dead-end inhibition studies indicate that cPRMT5 uses a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes. In total, these studies will guide PRMT5 inhibitor development and lay the foundation for studying how the activity of this medically relevant enzyme is regulated.

Wang M et al. (1999) Dev Biol "Competence and commitment of Caenorhabditis elegans vulval precursor cells."

Sternberg PW, Wang M

[Dev Biol, 1999]

Multipotent Caenorhabditis elegans vulval precursor cells (VPCs) choose among three fates (1 degrees, 2 degrees, and 3 degrees ) in response to two intercellular signals: the EGF family growth factor LIN-3 induces 1 degrees fates at high levels and 2 degrees fates at low levels; and a signal via the receptor LIN-12 induces 2 degrees fates. If the level of LIN-3 signal is reduced by a lin-3 hypomorphic mutation, the daughters of the VPC closest to the anchor cell (AC), P6.p, are induced by the AC. By expressing LIN-3 as a function of time in LIN-3-deficient animals, we find that both VPCs and the daughters of VPCs are competent to respond to LIN-3, and VPC daughters lose competence after fusing with the hypodermis. We also demonstrate that the daughters of VPCs specified to be 2 degrees can respond to LIN-3, indicating that 2 degrees VPCs are not irreversibly committed. We propose that maintenance of VPC competence after the first cell cycle and the prioritization of the 1 degrees fate help ensure that P6.p will become 1 degrees. This mechanism of competence regulation might have been maintained from ancestral nematode species that used induction both before and after VPC division and serves to maximize the probability that a functional vulva is formed.

Wang M et al. (1997) International C. elegans Meeting "COMPETENCE OF VPCS DURING C. ELEGANS VULVAL INDUCTION"

Wang M, Sternberg PW

[International C. elegans Meeting, 1997]

The C. elegans vulva is induced by a lin-3-encoded EGF-like growth factor produced by the anchor cell (AC). Each vulval precursor cell (VPC) is competent to respond to LIN-3 by adopting a either 1! or 2! fate during late L2 to early L3. The AC is necessary for wild-type vulval induction until about one hour before the VPCs divide. LIN-12 promotes differentiation of 2! cells. By expressing the EGF domain of LIN-3 under the control of an hsp16 promoter, we found that the VPCs lose their competence to respond to LIN-3 at or about their first division. To test the effect of activated LIN-12, we did a similar experiment in a lin-12 gain-of-function background. Based on observations of characteristic 1! lineages, expression of a 1! specific marker and adult vulval morphogenesis, we conclude that LIN-12 activity extends the end of the VPC competence after their first division. One model to explain this result is that lin-12, like its homologs, might promote cell proliferation. By stimulating the cell cycle, LIN-12 might drive VPCs along what is at first a common pathway, and thus remain competent to respond to LIN-3. Another model is that LIN-12 delays cell commitment to 3! fate, and maintains competence of uncommitted VPCs to respond to later signals. To examine the issue of whether the effect of LIN-12 in extending the window of VPC competence is due to an effect of LIN-12 stimulating the cell cycle, we are testing the effect of simply driving cell cycle using a cul-1 mutation.

Wang M et al. (2000) Development "Patterning of the C. elegans 1 degrees vulval lineage by RAS and Wnt pathways."

Wang M, Sternberg PW

[Development, 2000]

In C. elegans, the descendants of the 1 degrees vulval precursor cell (VPC) establish a fixed spatial pattern of two different cell fates: E-F-F-E. The two inner granddaughters attach to the somatic gonadal anchor cell (AC) and generate four vulF cells, while the two outer granddaughters produce four vulE progeny. zmp-1::GFP, a molecular marker that distinguishes these two fates, is expressed in vulE cells, but not vulF cells. We demonstrate that a short-range AC signal is required to ensure that the pattern of vulE and vulF fates is properly established. In addition, signaling between the inner and outer 1 degrees VPC descendants, as well as intrinsic polarity of the 1 degrees VPC daughters, is involved in the asymmetric divisions of the 1 degrees VPC daughters and the proper orientation of the outcome. Finally, we provide evidence that RAS signaling is used during this new AC signaling event, while the Wnt receptor LIN-17 appears to mediate signaling between the inner and outer 1 degrees VPC descendants.

Wang M et al. (1998) West Coast Worm Meeting "CELL CYCLE REGULATION OF C. ELEGANS VULVAL PRECURSOR CELL COMPETENCE"

Sternberg PW, Wang M

[West Coast Worm Meeting, 1998]

C. elegans vulval development allows analysis at a single cell level of the mechanisms that regulate the competence of cells to respond to inductive signals. By testing the response of C. elegans vulval precursor cells (VPCs) to the EGF family growth factor LIN-3 as a function of time, we demonstrate that VPCs lose competence at their first division. Activated LIN-12 extends this competence, as does delaying mitosis by hydroxyurea treatment. Promoting cell cycle progression by a cul-1 mutation or preventing cell fusion by overexpressing the HOM-C gene LIN-39 is also able to extend the end of VPC competence. We propose that VPC competence is regulated by the cell cycle, and there is a cell fate decision point during late S or early G2 of each cell cycle. VPCs can regain their competence by entering another cell cycle. Competence is progressively lost according to the number of cell cycles VPCs have undergone. In addition, our results suggest that LIN-39 activity may also be cell cycle-regulated. Activity of LIN-39 and/or other cell cycle regulated components might control VPC competence. Such a cell-cycle coupled mechanism may be commonly used in regulating the competence of precursor cells.

Wang M et al. (2021) PLoS Genet "Temperature regulates synaptic subcellular specificity mediated by inhibitory glutamate signaling."

Witvliet D, Wu M, Shao Z, Kang L, Wang M

[PLoS Genet, 2021]

Environmental factors such as temperature affect neuronal activity and development. However, it remains unknown whether and how they affect synaptic subcellular specificity. Here, using the nematode Caenorhabditis elegans AIY interneurons as a model, we found that high cultivation temperature robustly induces defects in synaptic subcellular specificity through glutamatergic neurotransmission. Furthermore, we determined that the functional glutamate is mainly released by the ASH sensory neurons and sensed by two conserved inhibitory glutamate-gated chloride channels GLC-3 and GLC-4 in AIY. Our work not only presents a novel neurotransmission-dependent mechanism underlying the synaptic subcellular specificity, but also provides a potential mechanistic insight into high-temperature-induced neurological defects.